WO2013162718A1 - Insulin secretion stimulated by humanin and analogs thereof - Google Patents

Abstract

Provided are methods of using humanin and humanin analogs to increase insulin secretion in a subject.

Description

I SULI SECRETION STIMULATED BY HUMANIN AND ANALOGS THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61/637,366, filed on April 24, 2012, the content of which is herein incorporated by reference into the subject application.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers AG027462 and AG035114 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention generally relates to use of humanin and humanin analogs, either alone or in combination with glucagon-like peptide 1 (GLP-1), to stimulate insulin secretion in a subject in need thereof.

BACKGROUND OF THE INVENTION

[0004] Throughout this application various publications are referred to in parenthesis. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.

[0005] Humanin (HN) is a 24 amino acid protein that has been shown to offer cyto- protection in response to a variety of insults such as ischemia, prion-induced apoptosis and chemical insults (37-40). It is thought to be transcribed from the 16S region of mitochondrial RNA, though multiple nuclear loci that code Humanin-like peptides have been identified. Recently, six additional peptides named SHLPs (small humanin-like peptides) have been identified to be encoded from open reading frames (ORFs) within the 16S rRNA. These SHLPS have been shown to have physiologically relevant roles in cell survival and metabolism. Though the exact site of origin of HN is not definitely established, biological effects of HN have been shown in multiple organ systems both in vivo and in vitro. In addition, molecular manipulations such as single amino acid substitutions have been shown to result in HN analogs with enhanced or altered biological activities. Recently it was shown that the HN analog HNG is more stable, and variations in biological activities were attributed to differences in stability in solution. In parallel to studies to establish the site of origin, studies have examined the role of HN and its analogs in various disease related situations. Many studies have shown a role for this peptide and its analogs in Alzheimer disease (AD) related insults in neurons, smooth muscle vascular cells (46) as well as in vivo models of AD. HNG has been shown to decrease infarct size following stroke, improve memory following AD- and scopolamine-related memory loss in rodent models, and provide cardio-protection in a model of myocardial ischemia-reperfusion.

[0006] In addition to a role in cyto-protection, HN and its analogs play a role in metabolism. HN and its potent analog HNGF6A have been shown to improve insulin sensitivity under hyperinsulinemic-euglycemic clamps. In addition, humanin has been shown to improve survival of beta cells and delay onset of diabetes in NOD mouse model of diabetes. Daily injections with HN for 6 weeks delayed the onset of diabetes in NOD mice, a model of type 1 diabetes by decreasing apoptosis in beta cells. The Humanin analog HNGF6A dramatically reduces blood glucose levels in the Zucker diabetic fatty (ZDF) rat, the rodent model of diabetes. With the fall in blood sugars, the ZDF rats exhibited changes in behavior, agitation that responded promptly after feeding. Interestingly, analysis of insulin levels in these animals demonstrated no decrease in insulin levels along with hypoglycemia.

[0007] The present invention addresses the need of subjects with abnormal insulin production or regulation.

SUMMARY OF THE INVENTION

[0008] The invention is directed to methods of increasing insulin secretion in a subject by administering humanin or an analog of humanin in an amount effective to increase insulin secretion.

[0009] The invention is also directed to methods of increasing insulin secretion in a subject by administering glucagon-like peptide 1 (GLP-1) and humanin or an analog of humanin in an amount effective to increase insulin secretion.

[0010] The invention also provides pharmaceutical compositions comprising glucagon- like peptide 1 (GLP-1) and humanin or an analog of humanin in an amount effective to increase insulin secretion in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1A-1B. Effects of HN on insulin secretion in BTC3 cells (A) with dose response (B). Cultured BTC3 cells treated with saline or HNGF6A in the presence of no (0 mM) or high (16mM) concentrations of glucose and insulin levels in the media were assayed by RIA after 2 hours of treatment. HNGF6A significantly increased insulin levels at high concentration of glucose (p< 0.05, A). B: Dose dependent effects of HNGF6A on insulin secretion were seen with maximal response at 50ng/ml of HNGF6A. No further increase in insulin levels was noted with increase in dose of HNGF6A (* p<0.0001).

[0012] FIG. 2. Time course effects of HN on insulin secretion in BTC3 cells. BTC3 cells were treated with 50 ng/ml of HNGF6A for 2, 5, 15, 30 and 60 min in the presence of 16mM glucose. Insulin levels in the media were assessed. Significant increase in insulin levels was seen at 60 min, though a trend towards increase in insulin levels is noticed at 15 and 30 minutes.

[0013] FIG. 3. Effects of HN on glucose sensing. BTC3 cells were transfected with GLUT2-GFP construct for 48 hours. Before the experiment, cells were cultured in glucose free media for 2 hours and then treated with 50 ng/ml of HNGF6A for 15 minutes at 16 mM glucose. There was enhanced GLUT-2 translocation to the plasma membrane in the presence of HNGF6A. Glucose phosphorylation rate was measured by glucokinase (GK) activity assay. BTC 3 cells were cultured in glucose-free media for 2 hours and then treated with scrambled peptide or HNGF6A for 5 or 15 min. Cytosolic fractions were used for GK acitivity assay as described herein in Materials and Methods. HNGF6A significantly increases GK activity.

[0014] FIG. 4A-4C. Effects of HN on intra-cellular ATP levels in BTC3 cells. BTC3 cells were treated with 50 ng/ml of HNGF6A at 16mM glucose concentration. Cellular ATP levels were measured at different time points after treatment with HNGF6A. As demonstrated, HNGF6A significantly increased intracellular ATP levels in BTC3 cells (p < 0.001, A), and this effect on ATP was abolished in the presence of AOA, an inhibitor of malate-aspartate shuttle (B). The decrease in ATP is associated with a decrease in insulin secretion (C).

[0015] FIG. 5A-5C. Effects of HN on intracellular calcium levels and role of diazoxide in the effects of HN. In the presence of HNGF6A, there was a steady increase in intracellular calcium levels around 50-60 minutes (Fig 5A). To define the role of KATP channels in the effects of HN on intracellular calicum and insulin release, BTC3 cells were treated with HN in the presence of diazoxide. HN was still able to increase insulin secretion in the presence of diazoxide, though the effect was attenuated (Fig 5B). Similarly, the intracellular calcium increased in response to FTNGF6A even in the presence of diazoxide suggesting that the increase in calcium levels are KATP channel independent (Fig 5C).

[0016] FIG. 6A-6B. Effects of FIN on insulin secretion in islets isolated from wild type and db/db diabetic mice. Islets isolated from 10-12 wk old C57BLK6/J mice and db/db mice (n=3 each) were treated with 50ng/ml of scrambled peptide or HNGF6A at 16mM glucose. There was a significant increase in insulin levels in the media at one hour treatment of HNGF6A compared to SP in wild type (A, p< 0.001) as well as db/db mice (B, p<0.001).

[0017] FIG. 7A-7E. Effects of HN on glucose stimulated insulin secretion in vivo. Young Sprague Dawley (SD, n=18) were subjected to 2 hr of hyperglycemic clamp (11 mM). Plasma glucose levels were acutely elevated to and maintained at 1 ImM (-200 mg/dL) using variable rates of glucose infusion. At 0 min, the HN group received an intravenous (IV) bolus of HNGF6A followed by maintenance dose for the duration of clamp (2 hours, total dose of 60 meg) while the control groups received saline. The glucose infusion rate (GIR) required to maintain a glucose level of l lmM was significantly higher in the HNGF6A treated group, (p< 0.0001). Data presented as time course (A) and average for the last hour of the clamp (B). This increase in GIR by HNGF6A was due to the significant increase in insulin levels shown as time course in (C) (p< 0.05) and average over the last one hour of the clamp (D). The increase is insulin is associated with tendency for an increase in C-peptide levels (E) and no change in calculated insulin clearance (data not shown).

[0018] FIG. 8A-8B. Humanin analog augments the effects of GLP-1 on glucose stimulated insulin secretion. Effects on insulin (A) and ATP (B) levels following treatment of βΤ03 cells with saline (sal), HNGF6A (F6A), Glucagon-like peptide 1 (GLP), and combined HNGF6A and Glucagon-like peptide 1 (F+G). *p<0.05; **p<0.01.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The invention is directed to a method of increasing insulin secretion in a subject comprising administering humanin or an analog of humanin to the subject in an amount effective to increase insulin secretion. In particular, insulin secretion is increased in the subject when blood glucose levels are elevated above normal.

[0020] The subject can have, for example, reduced production of insulin prior to administration of the humanin or humanin analog. The subject can have, e.g., an age- associated defect in insulin secretin. The subject can have, e.g., hepatic steatosis. The subject, e.g., can have, or be at risk for, type-1 or type-2 diabetes mellitus or maturity onset diabetes of the young (MODY).

[0021] The subject, e.g., can have, or be at risk for, insulin resistance. In subjects with insulin resistance, insulin has a reduced ability to lower blood sugars.

[0022] The treatments disclosed herein reduce the risk of inducing hypoglycemia in a subject, compared to treatment of a subject with, for example, sulfonylurea or glibenclamide, since in contrast to standard treatments, humanin and humanin analogs only increase insulin secretion in the presence of elevated glucose levels and not at normal blood glucose levels.

[0023] As used herein, humanin is the human peptide MAPRGF SCLLLLT SEIDLP VKRRA (SEQ ID NO: l) (GenBank AAK50430), or naturally- occurring vertebrate equivalents. Several analogs of humanin have been developed, some of them orders of magnitude more potent than humanin (e.g., 41-44). Examples include the analogs provided herein as SEQ ID NOs:2-25.

SEQ ID NO:2 - Humanin-like peptide from (45) amino acid sequence

MARRGFSCLL LSTTATDLPV KRRT;

SEQ ID NO:3 - Humanin analog F6A amino acid sequence

MAPRGASCLL LLT SEIDLP V KRRA;

SEQ ID NO:4 - Humanin analog S14G (HNG) amino acid sequence

MAPRGFSCLL LLTGEIDLPV KRRA;

SEQ ID NO:5 - Humanin analog AGA-HNG amino acid sequence

MAAGAFSCLL LLTGEIDLPV KRRA;

SEQ ID NO:6 Humanin analog HN17 amino acid sequence

PRGFSCLLLL T SEIDLP;

SEQ ID NO: 7 Humanin analog HNG 17 amino acid sequence

PRGFSCLLLL TGEIDLP;

SEQ ID NO:8 Humanin analog AGA-(C8R)HNG17 amino acid sequence

PAGASRLLLL TGEIDLP;

SEQ ID NO:9 Humanin analog colivelin amino acid sequence

SALLRSIPAP AGASRLLLLT GEIDLP;

SEQ ID NO: 10 combination of SEQ ID NO: l, 2, 3, 4, 5, 6, 7 and 8 (17mer) amino acid sequence (P/R/A)(R/A/G)(G/A)(F/A)S(C/R)LLL(L/S) T(S/T/G)(E/A)(I/T)DLP; SEQ ID NO: 11 - Humanin analog HNGF6A amino acid sequence

MAPRGASCLLLLTGEIDLPVKRRA;

SEQ ID NO: 12 Z-Humanin (Zebrafish) amino acid sequence

MAKRGLNCLPHQVSEIDLSVQKRI;

SEQ ID NO: 13 Humanin analog HNGF6AK21A amino acid sequence

MAPRGASCLLLLTGEIDLPVARRA;

SEQ ID NO: 14 C8A-HN (HNA) MAPRGFSALL LLTSEIDLPV KRRA;

SEQ ID NO: 15 D-Serl4-HN - MAPRGFS CLLLLTS *EIDLPVKRRA;

SEQ ID NO: 16 AGA-HNG - MAPAGASCLL LLTGEIDLPV KRRA;

SEQ ID NO: 17 AGA-(D-Serl4)- MAP AG AS CLLLLTS *EIDLPVKRRA;

SEQ ID NO: 18 HN AGA-(D-Serl4)- P AGAS CLLLLTS * EIDLP;

SEQ ID NO: 19 HN17 AGA-(C8R)- PAG AS RLLLLTGEIIDLP ;

SEQ ID NO:20 HNG17 EF-HN - EFLIVIKSMAPRGFSCLLLLTSEIDLPVKRRA;

SEQ ID NO:21 EF-HNA - EFLIVIKSMAPRGFSALLLLTSEIDLPVKRRA;

SEQ ID NO:22 EF-HNG - EFLIVIKSMAPRGFSCLLLLTGEIDLPVKRRA;

SEQ ID NO:23 EF-AGA-HNG - EFLIVIKSMAPAGASCLLLLTGEIDLPVKRRA;

SEQ ID NO:24 Colivelin - SALLRSPIPA-PAGASRLLLLTGEIDLP;

SEQ ID NO:25 P3R-HN - MARRGFS CLLLSTTATDLPVKRRT;

S* indicates D-Serine.

[0024] Preferred humanin analogs include ones that comprise of any of SEQ ID NO: 1- 25. Preferred humanin analogs include ones that consist essentially of any of SEQ ID NO: 1- 25, wherein the modification to any of SEQ ID NO: 1-25 does not decrease the ablility of the humanin analog to increase insulin secretion. Analogs of humanin be created, for example, by substitution of conservative amino acids into humanin or a known humanin analog. The skilled artisan could determine without undue experimentation the efficacy of a humanin analog in established humanin assays. Although the humanin or humanin analog can comprise non-peptide moieties, e.g., a fluorescent marker, it preferably consists entirely of a linear string of amino acids. More preferably, the humanin or humanin analog consists of less than about 50 amino acids. Even more preferably, the humanin or humanin analog consists of 17-50 or 17-26 amino acids.

[0025] Preferred humanin analogs include HNGF6A, which has the amino acid sequence MAPRGASCLLLLTGEIDLPVKRRA (SEQ ID NO: l 1). [0026] Preferred humanin or humanin analog comprise 17-50 or 17-26 amino acids and comprise the amino acid sequence of any of SEQ ID NO: 1 - SEQ ID NO:25.

[0027] As used herein, an amino acid sequence providing the designation (x/y), as in SEQ ID NO: 10, indicates that either amino acid x or amino acid y can be used at the indicated position. Thus, the amino acid sequence (p/r/a)(r/a/g)(g/a)(f/a)s indicates that the first amino acid can be a proline, alanine or glycine; the second amino acid can be arginine, alanine or glycine, the third amino acid can be glycine or alanine, the fourth amino acid can be phenylalanine or alanine, and the fifth amino acid must be serine.

[0028] The present methods can be used with any mammalian species. The subject is preferably a human.

[0029] Humanin or humanin analog can be administered directly to the subject. Alternatively, the humanin or humanin analog can be administered by administering a vector encoding humanin or a humanin analog to the subject such that humanin or the analog is expressed from the vector. Such vectors can be prepared for any given application without undue experimentation.

[0030] The humanin or humanin analog is preferably administered in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" it is meant a material that (i) is compatible with the other ingredients of the composition without rendering the composition unsuitable for its intended purpose, and (ii) is suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are "undue" when their risk outweighs the benefit provided by the composition. Non-limiting examples of pharmaceutically acceptable carriers include, without limitation, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, microemulsions, and the like.

[0031] The humanin or humanin analog can be formulated without undue experimentation for administration to a mammal, including humans, as appropriate for the particular application. Additionally, proper dosages of the compositions can be determined without undue experimentation using standard dose-response protocols.

[0032] The humanin or humanin analog can easily be administered parenterally such as for example, by intravenous, intraperitoneal, intramuscular, intrathecal or subcutaneous injection. Parenteral administration can be accomplished by incorporating the compounds into a solution or suspension. Such solutions or suspensions may also include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Parenteral formulations may also include antibacterial agents such as for example, benzyl alcohol or methyl parabens, antioxidants such as for example, ascorbic acid or sodium bisulfite and chelating agents such as EDTA. Buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be added. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

[0033] Rectal administration includes administering the humanin or humanin analog, in a pharmaceutical composition, into the rectum or large intestine. This can be accomplished using suppositories or enemas. Suppository formulations can easily be made by methods known in the art. For example, suppository formulations can be prepared by heating glycerin to about 120° C, dissolving the composition in the glycerin, mixing the heated glycerin after which purified water may be added, and pouring the hot mixture into a suppository mold.

[0034] Transdermal administration includes percutaneous absorption of the composition through the skin. Transdermal formulations include patches (such as the well- known nicotine patch), ointments, creams, gels, salves and the like.

[0035] The present invention includes nasally administering to the mammal a therapeutically effective amount of the humanin or humanin analog. As used herein, nasally administering or nasal administration includes administering the compound to the mucous membranes of the nasal passage or nasal cavity of the patient. As used herein, pharmaceutical compositions for nasal administration of the compound include therapeutically effective amounts of the compound prepared by well-known methods to be administered, for example, as a nasal spray, nasal drop, suspension, gel, ointment, cream or powder, or using cubosomes (47). Administration of the compound may also take place using a nasal tampon or nasal sponge.

[0036] Where the compound is administered peripherally such that it must cross the blood-brain barrier, the compound is preferably formulated in a pharmaceutical composition that enhances the ability of the compound to cross the blood-brain barrier of the mammal. Such formulations are known in the art and include lipophilic compounds to promote absorption. Uptake of non-lipophilic compounds can be enhanced by combination with a lipophilic substance. Lipophilic substances that can enhance delivery of the compound across the nasal mucus include but are not limited to fatty acids (e.g., palmitic acid), gangliosides (e.g., GM-1), phospholipids (e.g., phosphatidylserine), and emulsifiers (e.g., polysorbate 80), bile salts such as sodium deoxycholate, and detergent-like substances including, for example, polysorbate 80 such as Tween™, octoxynol such as Triton™ X-100, and sodium tauro-24,25-dihydrofusidate (STDHF). The humanin or humanin analog can also be combined with micelles comprised of lipophilic substances. Such micelles can modify the permeability of the nasal membrane to enhance absorption of the compound. Suitable lipophilic micelles include without limitation gangliosides (e.g., GM-1 ganglioside), and phospholipids (e.g., phosphatidylserine). Bile salts and their derivatives and detergent-like substances can also be included in the micelle formulation. The compound can be combined with one or several types of micelles, and can further be contained within the micelles or associated with their surface.

[0037] The humanin or humanin analog used in the methods of the invention may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salts should be both pharmacologically and pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare the free active compound or pharmaceutically acceptable salts thereof. Pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicyclic, p-toluenesulfonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzenesulphonic. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

[0038] Humanin or humanin analog can be administered peripherally to the mammal. A preferred peripheral administration for these methods is parenteral administration. Most preferably, the humanin or humanin analog is administered intravenously to the mammal.

[0039] In the methods disclosed herein glucagon-like peptide 1 (GLP-1) can be administered to the subject in combination with humanin or an analog of humanin. As described herein, combined treatment with GLP-1 and humanin or an analog thereof can produce a synergistic increase in insulin secretion.

[0040] The invention also provides a pharmaceutical composition comprising glucagon-like peptide 1 (GLP-1) and humanin or an analog of humanin in an amount effective to increase insulin secretion in a subject. Preferred humanin analogs include analogs comprising 17-50 amino acids or 17-26 amino acids comprising the amino acid sequence of any of SEQ ID NO: l - SEQ ID NO:25. The human analogs can comprise or consist of or consist essentially of the amino acid sequence of any of SEQ ID NO: l - SEQ ID NO:25. Preferred humanin analogs include HNGF6A, which has the amino acid sequence MAPRGASCLLLLTGEIDLPVKRRA (SEQ ID NO: 11). As used herein, a humanin analog that "consists essentially of a specified sequence means that the additions to the specified sequence do not decrease the effectiveness of the specified sequence in increasing insulin secretion.

[0041] The invention also provides humanin and humanin analogs for use in a method of increasing insulin secretion in a subject.

[0042] This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.

EXPERIMENTAL DETAILS

Materials and Methods

In vitro studies

[0043] Cell culture: Mouse pancreatic-β cells (BTC3), kindly provided by Dr. Norman Fleischer, were maintained in DMEM containing 10% heat- inactivated FBS, D-glucose (4500 mg/L), and glutamine (4mM) at 37°C in a humidified environment with 5% C02. The initial studies probing the effects of HNGF6A were done at glucose concentrations of 0 mM, 5 mM and 16 mM glucose levels. Subsequent studies were all done at 16 mM glucose levels. For the dose response study, BT3C Cells were grown to 70% confluence in 60mm dishes and treated with either 24-aa scrambled peptide (SP, Sequence: FRGGETRARAMPLIDLSPLCLLKV (SEQ ID NO:26)) or varying doses of HNGF6A between 5-500 ng/ml (Sequence: MAPRGASCLLLLTGEIDLPVKRRA (SEQ ID NO: 1 1), Genemed Custom peptide synthesis, Texas) and insulin in the media was assessed at 2 hours. For the time course study, BT3C Cells were treated for the indicated times with 50ng/ml of either scrambled peptide or HNGF6A. Insulin levels were assayed in the media using the Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem Inc.).

[0044] GLUT2 Translocation: BTC3 cells were seeded on cover slips in a 6-well plate one day before transfection. Cells were transfected with 2 ug of GLUT2-GFP construct using Lipotransfectamine 2000 according to the manufacturer's instructions (Invitrogen). Cells were incubated in media without glucose for overnight and then treated with 50ng/ml HNGF6A or SP at 16 mM Glucose for 15 min. After 2 washes with cold PBS, cells were fixed using 4% formaldehyde for 20 min. Images were taken using Leica SP2 Confocal microscopy. GLUT2 translocation rate was quantified using ImageJ software.

[0045] Glucokinase activity assay: Glucose phosphorylation rate was measured by a well-established fluorimetric assay (ref). Glucose is phosphorylated by glucokinase and hexokinase in a reaction with ATP. The product, glucose-6-phosphate can be oxidize to 6- phosphogluconate by glucose-6phosphate dehydrogenase in the presence of NAD+, and meanwhile, NAD⁺ is reduced to NADH. The increase of NADH can then be monitored by recording the increase in fluorescence at 460 nm with excitation at 340 nm. BTC3 cells were washed twice in glucose-free KRHB buffer after treated with 50 ng/ml SP or HNGF6A for different time points and then homogenized in 500 ul of cold lysis buffer (10 mM HEPES pH 7.4, 250 mM sucrose, 2mM EDTA, ImM DTT, 1.5 mM MgC12, and 10 mMKCl) by going through 22 gate needle for 10 times. The soluble cytosolic fractions collected from 100,000X g centrifugation were used for kinase assay. In brief, 250 μg soluble protein of each samples was added to 100 μΐ assay buffer (50 mM HEPES, pH 7.7, 100 mM KC1, 10 mM MgC12, 15 mM beta-Mercaptoethanol, 0.5 mM NAD+, 5 mM ATP, 10 μg/ml G6PDH and 0.05% BSA) containing 0.5 mM or 50 mM glucose and incubated at 37 C for 1 hour. A reaction mix with cytosol protein, glucose and assay buffer without ATP was also created for background subtraction. Relative glucokinase activity was calculated by subtracting values of Hexokinase (collected from 0.5 mM glucose reactions) from values of total kinases (collected from 50 mM glucose reactions).

[0046] Bioluminescent Assay for Intracellular ATP: BT3C Cells were grown to 70% confluence in 60mm dishes and treated for the indicated time with 50ng/ml of either the 24- aa SP or HNGF6A. Media was aspirated and the dishes rinsed briefly with ice-cold PBS. Cells were scraped into ice-cold 350μ1 of 85mM sodium citrate. A 200μ1 aliquot of the dispersed cells was brought to 2.3%TCA in order to rapidly extract the ATP and inactivate cellular enzymes as described previously (Lundin A, et ah, 1986) An additional aliquot of the dispersed cells was diluted 1 : 1 with Trypan blue and the cells/ml determined using a Countess® automated cell counter (Invitrogen). The extracted lysate was diluted 1 : 10 in Tris- Acetate buffer, pH 7.75, containing 2mM EDTA, incubated at RT for 30 minutes, boiled for 3 minutes and placed on ice. This product and a series of ATP standards were added, in duplicate, to a 96-well plate and assayed for ATP with an Adenosine 5 '-triphosphate (ATP) Bioluminescent Assay Kit (Sigma-Aldrich, FL-AA) and a LUMIstar ΟΡΤΓΜΑ- Luminescence Microplate Reader with an injector (BMG LABTECH). Picomoles of ATP/ml were determined and normalized to cells/ml.

[0047] Cells were also studied in the presence of aminooxyacetate (AOA), malate- aspartate shuttle inhibitor; treatment with this inhibitor significantly decreases pyruvate production and effectively reduces the size of the alpha-ketoglutarate pool in rapid exchange with the TCA cycle. Insulin levels were assessed at one hour in the presence or absence of FTNGF6A after pre-incubation for 2 hours with AOA.

[0048] One aliquot of the media in which BTC3 cells were grown and treated for the above bioluminescent assay for intracellular ATP was collected and stored at -80°C for insulin estimation. The initial range of insulin levels in representative samples was assessed with the wide range assay as described in the Ultra Sensitive Mouse Insulin ELISA Kit Instructions (Crystal Chem Inc.). The same kit was used to determine the all sample insulin concentrations using the high range assay and normalizing the results (pg/ml) to cell counts from each plate (cells/ml).

[0049] Calcium kinetic measurements: BT3C (5xl0⁴) cells were seeded in 96 well black plates with clear bottoms (353948, BD Falcon, NJ) and allowed to attach overnight at 16mM glucose. The cells were incubated with 25 μΜ Fura-2/AM diluted in PBS (F1221, Invitrogen Molecular Probes) for 60 min at 37°C. The wells were rinsed with PBS three times and then the plate placed on ice and exposed to PBS containing either SP or FTNGF6A (50ng/ml) with and without diazoxide (depending of the study group). The cells were then transferred to SpectraMaxMF⁶ temperature-regulated chamber at 37°C (Molecular Devices Ca) without washing and photometric data for [Ca²⁺] was generated by exciting cells at 340 and 380nm and measuring emission at 510nm every 5 minutes for two hours. An intracellular calibration was performed with each experiment by determining the fluorescence ratio (340:380) in the presence of Ca-free lOmM K₂EGTA buffer (R_min) and lOmM CaEGTA buffer containing 10μΜ ionomycin (R_max) (C-3008, Calcium Calibration Buffer Kit #1, Invitrogen Molecular Probes). The mean [Ca²⁺] was determined from 12 wells using the following equation: [Ca²⁺]= KaQ (R-R_mi_n)/(R_max-R), where R represents the fluorescence intensity ratio

λ1(340ηιη) and λ2(380ηιη) are the fluorescence detection wavelengths for ion-bound and ion- free indicators; ¾ is the Ca²⁺ dissociation constant and equals 0.14 μΜ (Fura and Indo Ratiometric Calcium Indicators, Invitrogen Molecular Probes); and Q is the ratio of F_mi_n to F_max at λ2 (380nm). During the two hours of Ca estimation, the cells were in glucose free medium. The experiments were repeated in the presence of 16 mM glucose through the study (even during the estimation of Ca).

[0050] Mouse pancreatic islets: Islets from 25-30 g male C57/Blk 6 (wt) and db/db (diabetic) mice were isolated using standard collagenase disgestion as described (1) and cultured overnight in RPMI medium supplemented with 10% FBS plus antibiotics. Prior to experiments, islets were transferred to MilliCell-PCF culture plate filter inserts (Millipore) at a density of -10-20 islets/insert. The inserts were placed within individual wells of a 24-well cell culture plate, and each well was filled with 1 ml of DME, 5 mM glucose. After a 6 hour preincubation at 5 mM glucose, the inserts were transferred to new wells containing 0.5 ml volume of media and islets were challenged with glucose (5 mM or 16mM) and varying concentrations of FTNGF6A as indicated. In some experiments, the effects of 16 mM glucose plus pyruvate (2mM) in the presence and absence of FTNGF6A were also tested. Media was collected from beneath inserts; islets were floated by a rapid applications of 0.5 ml of PBS added to the inserts. Islets were pelleted and then lysed by sonication as described (2). An antiprotease cocktail containing aprotinin (1 mU/ml), leupeptin (0.1 mM), pepstatin (10 ~M), EDTA (5 mM), and diisopropylfluorophospbate (1 mM) was added to the collected media and cell lysates. All samples were spun in a microfuge to remove debris before electrophoresis. Aliquots of cell lysates were subjected to SDS PAGE and immunoblotting. Immunoblots were probed for insulin and actin content using guinea pig anti-insulin (Linco Research) and rabbit anti actin (Sigma) antibodies. Insulin levels in the media were measured by ELISA as described above for BTC3 cells and densitometric analysis of immunoblots was used to normalize insulin levels in media. The repeatability of all findings was confirmed by performing each experiment a minimum of three times.

[0051] Humanin analog augments the effects of GLP-1 on glucose stimulated insulin secretion. TC3 cells, a well characterized, glucose responsive murine beta cell line, were grown in 16mM glucose in a 24-well plate overnight. Cells were treated with FTNGF6A (19 nM, dose demonstrated to have maximal effect on insulin secretion), glucagon-like peptide- 1 (GLP-1) (10 nM, dose shown to have maximal effects on insulin secretion in vitro), and the combination of FTNGF6A and GLP-1 at submaximal doses of 9.5 nM and 5 nM, respectively, for 1 hour. Insulin levels in media were determined using an Ultrasensative mouse insulin Elisa kit (Crystal-chem, MO). The experiment was repeated 3 times. In a separate experiment, under similar conditions, ATP levels were measured with an Adenosine 5'- triphosphate (ATP) Bioluminescent Assay Kit (Sigma-Aldrich, MO).

In vivo studies

[0052] Hyperglycemic clamp in vivo: Young (3 mo old) male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were used for this study. Rats were housed in individual cages and were subjected to a standard light (6:00 AM to 6:00 PM)-dark (6:00 PM to 6:00 AM) cycle. All rats were fed ad libitum using regular rat chow that consisted of 64% carbohydrate, 30% protein, and 6% fat with a physiological fuel value of 3.3-kcal/g chow. Indwelling catheters were placed in the right internal jugular vein and in the left carotid artery (6, 23). The venous catheter was extended to the level of the right atrium, and the arterial catheter was advanced to the level of the aortic arch. Recovery was continued until body weight was within 3% of the pre-operative weight (-4-6 days). Hyperglycemic Clamp studies, the gold standard to measure insulin secretion capacity were performed in awake, unstressed, chronically catheterized rats (5, 6, 22, 23).

[0053] Hyperglycemic clamp protocol: All rats (n=18) were subjected to a 2 hour moderate hyperglycemia (~l lmM) as previously described. Briefly, 25% glucose was infused intravenously to raise the plasma glucose concentration acutely to -l lmM. The glucose infusion rate was then varied to maintain the plasma glucose concentration at this level for 2 hours. A total dose of 60 μgram of HNGF6A was administered per animal, a third was given as a bolus to and the remaining HNGF6A was administered as a continuous infusion at the rate of the 0.07mg/kg/hr over 2 hours. Plasma samples for insulin were obtained at 10-min intervals and C-peptide at 30 min intervals throughout the study. At the end of the clamp study, rats were sacrificed using lOOmg pentobarbital sodium/kg body wt IV. The study protocol was reviewed and approved by the Animal Care and Use Committee of the Albert Einstein College of Medicine.

[0054] Terminology and calculations: The area under the curve (AUC) of the first phase insulin response was calculated by the trapezoid rule using 0, 2, 4, 6, 8, 10 min samples with the formula [(i 0 + i 2)12 x 0.5 + (i 2 + i 4)/2 x 0.5 + (i 4 + i 6)/2 x 0.5 + (i 6 + i 8)/2 x 0.5 + (i 8 + i 10)/2 x 0.5]. Insulin and C-peptide levels were averaged over the last one hour of the clamp. [0055] Analytical procedures: Plasma glucose was measured by the glucose oxidase method (Glucose Analyzer II; Beckman Instruments, Inc., Palo Alto, CA) and plasma insulin was measured by radioimmunoassay using rat insulin standards in the invivo experiements.

[0056] Statistical analysis: All values shown are expressed as means ± SE. Statistical Analyses were performed using analysis of variance in multiple comparisons. When the main effect was significant, a two-tailed post hoc test (Bonferroni) was applied to determine individual differences between means. A P value < 0.05 was considered to be statistically significant. All statistical analyses were performed using Excel and Graph pad for Windows.

Results

[0057] Humanin increases GSIS in a dose dependent manner in vitro: Glucose metabolism is the primary initiator of insulin exocytosis and upon stimulation by glucose, β cells secrete insulin. To test if HN regulated insulin secretion, basal and glucose-induced insulin release were characterized in a glucose sensitive beta cell line, BTC3 (3, 4) (generous gift from Dr. N. Fleischer, Albert Einstein College of Medicine, New York, NY). BTC3 cells were incubated in the presence of no glucose, 5 mM or 16 mM glucose, and insulin levels assessed in the media at 2 hours. A significant increase in insulin secretion was observed in the presence of 16 mM glucose while there was no significant increase in insulin at the 0 mM (Fig 1A) or 5 mM glucose (data not shown). BTC3 cells were then incubated at 16 mM with the addition of varying concentrations of FTNGF6A or a 24-aa SP of FTNGF6A. Dose dependent augmentation of insulin release was observed (Fig IB). Maximal effects were reached at 50ng/ml (Fig IB), and this FTNGF6A concentration was used in all subsequent experiments unless noted otherwise.

[0058] Humanin increases GSIS in a time dependent manner in vitro: GSIS occurs in a biphasic manner: an early, first-phase insulin release occurs during the first few minutes of glucose stimulus followed by a prolonged, second-phase of GSIS (1, 2). To better understand the role of FTNGF6A on the different phases of insulin secretion, the time dependent effects of FTNGF6A on insulin release were studied. BTC3 cells were incubated with 50mg FTNGF6A/ml (the dose that produced the maximal effect on insulin secretion) for varying time points. Insulin was assessed at 5, 15, 30, 60 and 120 min at both 5mM and 16mM glucose concentrations. FTNGF6A significantly increased insulin release in the presence of 16mM glucose; Robust HNGF6A augmented insulin secretion was first observed only after 60 minutes of incubation (Fig. 2). There was no stimulation of insulin secretion at basal glucose concentrations (data not shown).

[0059] Humanin increases translocation of GLUT-2 and enhances giucokinase activity in BTC3 cells: BTC 3 cells were transfected with GLUT2-GFP for 48 hours. The cells were exposed to 16mM glucose in the presence of SP or FTNGF6A for 15 mins. GLUT 2 tranlocates to the surface of the BTC3 cells in the presence of 16mM glucose. This translocation is significantly enhanced in the presence of HNGF6A (data not shown). Since giucokinase is the rate limiting step in further metabolism of the glucose that is taken up by the beta cell, the activity of the enzyme giucokinase was studied in the presence of FTNGF6A at 5 and 15 minutes post treatment. Giucokinase activity is significantly enhanced by FTNGF6A at both the 5 minutes and 15 minute time points (Fig. 3).

[0060] HN induced GSIS is linked to increase in cellular ATP production: Increased plasma glucose levels initiate increased glucose uptake, glycolysis, and mitochondrial metabolism by the β-cell. Higher rates of glycolysis and mitochondrial metabolism lead to higher ATP resulting in a series of events mediated by a closure of the ATP-dependent potassium channels (KATP), influx of Ca²⁺ and activation of exocytosis of insulin-containing granules (5-10). Several reports in the literature have convincingly demonstrated that glucose controls insulin secretion by a dual, K_ATP dependent and independent mechanism; the triggering Ca2+ signal is necessary for both phases of glucose-induced insulin secretion (11, 12). The time line of the effects of H GF6A on ATP levels and calcium flux in the beta cell was systematically explored. BTC3 cells were exposed to H GF6A in 16 mM glucose, and cellular Ca²⁺ and ATP levels were measured over time. In the presence of glucose, HNGF6A stimulated both an increase in ATP production (Fig. 4A) and cellular Ca²⁺ accumulation (Fig. 5A). The most pronounced increase of HNGF6A dependent cellular ATP levels required a prolonged, 60 minute incubation. Consistent with the effects seen on ATP levels, maximal UN stimulated Ca²⁺ accumulation was significantly delayed with a significant UN-dependent rise in Ca²⁺ observed after the 60 minute time period (Fig. 5A). There was no significant increase in ATP levels in BTC3 cells at 5 mM glucose.

[0061] Humanin 's role on GSIS is mediated by effects on mitochondria: ATP is generated by both cytosolic and mitochondrial reactions and each of these discrete sources of ATP are thought to play an important role in regulating insulin secretion (6, 13-23). Mitochondrial ATP generation in particular, plays a key role in coupling glucose metabolism with insulin secretion (24, 25). The present observations suggested the possibility that HNGF6A's time-delayed augmentation of insulin secretion using a fuel-only stimulus is mediated by mitochondrial substrate cycling. To explore the potential mechanism by which FTNGF6A may regulate insulin secretion, the next set of experiments were done in the presence of amino-oxyacetate (AOA), a well-characterized inhibitor of the malate-aspartate shuttle (14). In these experiments, BTC3 cells were incubated in the presence of the amino- oxyacetate (AOA) for 2 hours in the presence of FTNGF6A or scrambled peptide. As anticipated, AOA dampened cellular ATP levels irrespective of FTNGF6A addition (Fig. 4B). Worthy of note were the effects on hormone secretion: AOA specifically ablated FTNGF6A dependent insulin release (Fig. 4C).

[0062] Effects of HN on intracellular calcium and insulin secretion are independent of KATP channel. Calcium flux into the beta cell and subsequent insulin release are mediated through closure of KATP channel. In order to identify the specific role of KATP channel in the role of UN in intracellular calcium fluxes and insulin release, BTC3 cells were treated with FTNGF6A in the presence of Diazoxide. Diazoxide keeps the KATP channel open and prevents calcium flux through voltage-dependent calcium channels. FTNGF6A increased insulin secretion compared to SP in the presence of Diazoxide, but the effect was attenuated (Fig 5B). FTNGF6A increased intracellular calcium levels even in the presence of Diazoxide (Fig 5C).

[0063] Humanin augments insulin secretion in isolated islets of wild type and db/db mice. When exposed to brief or extended glucose stimulation, healthy islets respond with a robust release of insulin. The pathways that initiate, amplify and inhibit insulin secretions have been well studied. Specifically, low activity of NADH shuttles in ?-cells has been found in aging (33, 34) and in models of Type II diabetes (35) leading to decreased insulin secretion. Islets isolated from wild type and db/db mice were incubated with FTNGF6A and insulin secretion was assessed at 1 and 2 hours. At stimulating glucose concentration (16 mM), the insulin release of islets freshly isolated from wild type mice was significantly augmented in the presence of HNGF6A. Some variability was observed in the response of islets and in most cases, insulin release from db/db islets relative to wild type controls, was reduced. Nevertheless, exposure of islets to glucose plus FTNGF6A caused an increase of insulin release in both control and diabetic islets (Fig. 6A, 6B). [0064] HN induced insulin secretion is linked to the increase in pyruvate levels: To exclude the contribution of Glut-2 and glucokinase, the two critical proteins that control the rate of glucose metabolism in HNGF6A stimulated insulin secretion, islets were incubated in the presence of pyruvate. Addition of pyruvate bypasses the flux through glycolysis and provides the substrate leading to TCA cycle and ATP generation. The interesting finding was that the addition of HNGF6A over pyruvate did not result in further increment in insulin secretion (over pyruvate alone) suggesting that the effects of HNGF6A are secondary to increased flux through glycolysis, increased pyruvate and subsequent increase flux through the TCA cycle (10.3 ± 0.2 vs. 11.2 ± 0.05 in pyruvate+ scrambled peptide vs. Pyruvate + FTNGF6A respectively). This is consistent with the observation in BTC3 cells using AOA, a malate-aspartate shuttle inhibitor that specifically lowers pyruvate levels.

[0065] Humanin analog augments the effects of GLP-1 on glucose stimulated insulin secretion. As expected, GLP-1 increased glucose-stimulated insulin secretion (GSIS) from TC3 cells. At the concentrations used, HNGF6A-induced increase in GSIS was similar to GLP-1 (SEM control 857.43+/- 180.99 vs. HNGF6A 2091+/- 406.3126.37 p value 0.2, vs. GLP-1 1460.03+/- 136.401 p value 0.03). Interestingly, there was a synergetic effect on GSIS in the presence of both HNGF6A and GLP-1 (SEM combination 3348.63+/- 366.12, p value combinations vs. saline 0.0004, combination vs.HNGF6A 0.04, combination vs. GLP-1 0.0005) (Figure 8A).

[0066] Consistent with the insulin levels, while HNGF6A alone and GLP-1 alone increased ATP levels compared to controls ( SEM control 153.29+/- 14.42, HNGF6A 242.53+/ 26.377 p value 0.04, GLP-1 303.74+/- 13.99, p value 0.1), the increase was significantly higher in wells treated with both HNGF6A and GLP-1 ( SEM 429.85/6.98, p value 0.001) (Figure 8B).

[0067] Humanin increases insulin secretion in vivo (Figure 7): To determine the extent to which HN dependent augmentation on insulin secretion observed in in vitro studies could be replicated in vivo, systemic administration of HN on insulin secretion was tested using whole animals. The role of HNGF6A was assessed in vivo using the gold standard hyperglycemic clamps. Groups were matched in terms of age, body weight, basal glucose, insulin (data not shown). The presence of CNTFR-a and Gp 130, two components of the proposed receptor mediating the effects of HN, was confirmed in the rat pancreas by western blot. During the in vivo hyperglycemic clamp, glucose level was clamped at 1 lmM in all the rats. The coefficient of variation of glucose levels during the clamps were 2.98% and 2.55% in HNGF6A and control groups respectively. Animals that received HNGF6A needed a significantly higher glucose infusion rate (GIR) during the clamp to maintain euglycemia (55.1 ± 0.7 vs. 42.5 ± 1.0 mg/kg/min in HNGF6A vs. controls respectively, p< 0.0001). The GIR was statistically different from ~60 min in FTNGF6A treated animals compared to saline controls and remained significantly different throughout the duration of the clamp. The difference in GIR was attributed to significant differences in insulin secretion between both groups as glucose-induced insulin secretion was significantly higher in the HNGF6A treated animals from ~40 minutes till the end of the clamp. When insulin levels were averaged during the last one hour of the clamp after steady state was achieved, the insulin level in the FTNGF6A treated animals were twice that of control groups (9.9 ± 2.0 vs. 4.61 ± 0.8 ng/ml in FTNGF6A vs. controls, p < 0.001). The area under the curve for C-peptide tended to be higher in HNGF6A treated animals (2575.7 ± 360.36 vs. 1871.26 ± 300.63 in HNGF6A vs. controls respectively, p =0.14) compared to controls. There were no differences in the insulin clearance between the two groups (data not shown).

[0068] Area under the curve (AUC) for first phase insulin response during the initial 10 min of the clamp tended to be higher in FTNGF6A treated animals, but this did not reach statistical significance (46.5 ± 9.1 vs. 35.2 ± 5.6 ng/ml.min in HNGF6A vs. control respectively, p=0.32). UN's amplification of glucose-induced insulin secretion appeared to be due to a direct effect on the islet. Moreover, in support of the in vitro studies, FTNGF6A influenced insulin secretion in vivo by primarily targeting amplification of the second phase of insulin release.

Discussion

[0069] The present experiments demonstrate for the first time the effects of a UN analog FTNGF6A on insulin secretion in vivo and in vitro. Using stable cell lines and islets, dose effect, time course and potential mechanisms through which UN increases insulin secretion were demonstated. Using islets isolated from diabetic mice, this effect was demonstrated to be robust in both wild type and diabetic mice.

[0070] Defects in both insulin secretion and action are integral components of diabetes. Drugs used in diabetes can be either insulin sensitizers or insulin secretagogues. There are insulin sensitizers like metformin that do not increase insulin secretion, and insulin secretagogues such as sulfonylurea that do not increase insulin sensitivity. There are very few drugs that can do both, an example of which are GLP-1 analogs and DPP-4 inhibitors (that increase GLP-1 levels).

[0071] Insulin sensitizing effects of HN have previously been demonstrated (42, 44). The effects of HN on glucose stimulated insulin secretion demonstrated here are independent of the effects of HN on insulin action. These effects are also distinct from the effects shown with chronic HN treatment in NOD mice, where HN improved glucose homeostasis and delayed onset of diabetes through decrease in apoptosis and improvement in beta cell survival. The ability of insulin to effect both insulin action and secretion are unique as few currently available therapeutic options to treat diabetes have beneficial effect on both aspects of glucose metabolism. In that regard, HN is similar to IGF-1 and GLP-1.

[0072] In vivo studies described here were done with a single dose of HN and the dose of HN was chosen based on previous studies. The effects of HNGF6A on insulin secretion were seen at -40 minutes under hyperglycemic clamp conditions and persisted throughout the duration of the study. The AUC of the C-peptide tended to be higher in HNGF6A treated group. This, in combination with the lack of differences in the insulin clearance rates between the groups, suggests that the higher levels of insulin are due to increased release of insulin from the beta cells. In earlier studies with HN, HNGF6A suppresses free fatty acids (FFA) levels, demonstrating that the effects of HN on insulin secretion in vivo are not mediated through an increase in FFA.

[0073] The present in vitro studies with BTC3 cells, an exclusive beta cell line, and islets isolated from mice helped to overcome the limitation of a single dose in vivo study and help understand the dose response and time course. The studies in the BT3C cells not only confirmed the role of HNGF6A in increasing insulin secretion, they also show that the effects of humanin are dose dependent with maximal effects seen at 50ng/ml of HNGF6A.

[0074] The earliest time point with a significant increase in insulin secretion was at around 40-50 minutes in vivo and at one hour in in vitro. The slow kinetics observed point to the possibility that HNGF6A-enhanced insulin release may be mediated through a time- delayed, K_ATP channel-independent mechanism and is mediated through substrate metabolism. This is consistent with the lack of a significant first phase response of insulin in the in vivo studies. The delayed response is also consistent with the time when significant increases in cellular ATP levels were observed in vitro, thus linking increase in cellular ATP in response to HNGF6A to observed effects on insulin secretion. Increases in ATP levels with HN have been reported in muscle cells.

[0075] The effects of HNGF6A on ATP links mitochondrial metabolism to the effects on insulin secretion. Mitochondria are the major source of ATP and are at the heart of the 'glucose metabolism- insulin secretion' coupling, linking glucose recognition to insulin exocytosis (28; 29). Mitochondria serve as both recipients of glucose derived metabolites and generators of signals (ATP) that increase insulin secretion; decreased glycolytic flux and uncoupling of mitochondrial oxidative phosphorylation results in impaired nutrient stimulated insulin secretion (28). There is also compelling evidence to show that ATP is a key factor coupling mitochondrial metabolism to insulin secretion (28) and functional KATP channels are required for GSIS. People with Mitochondrial DNA mutations demonstrate impaired GSIS and higher T2DM (30-33) highlighting the role of mitochondria on insulin secretion. Several reports support the notion that cytosolic NADPH may ultimately couple mitochondrial activity with downstream events leading to insulin secretion (27-29).

[0076] The effects of HNGF6A on GLUT2 tranlsocation, GK-the key rate limiting enzyme point to increased glucose utilization in the beta cell in response to FTNGF6A. Unlike myocytes and hepatocytes, β-cells cannot accommodate increased glucose uptake under hyperglycemic conditions by storing the excess glucose as glycogen or eliminate it as lactate. Substrate cycling allows glycolytic and mitochondrial fluxes to increase in proportion to circulating concentrations of glucose and this increase in the concentrations of several Kreb's cycle intermediates, generates mitochondrial NADH reducing equivalents which are then transferred to cytosolic NADPH (reviewed in (26). The NADH shuttle system is composed essentially of the glycerophosphate and the malate/aspartate shuttles (30). In β-celh however, the malate/aspartate shuttle appears to play a predominant role as insulin release, glucose metabolism and ATP levels in glycerol phosphate dehydrogenase deficient islets remain normal (31, 32). AOA is an inhibitor of malate-aspartate shuttle. Treatment with AOA has been shown to significantly decrease pyruvate, reduce the size of the alpha-ketoglutarate pool in rapid exchange with the TCA cycle and decrease ATP production (36) in human leukemia cell line. The observed loss of increase in ATP and insulin secretion with HNGF6A in the presence of specific malate-aspartate shuttle inhibitor, AOA highlights the role of mitochondria and TCA flux in mediating the effects of HNGF6A. Along with the data on isolated islets with pyruvate, these studies demonstrate that the effects of HN may be mediated through an increase in pyruvate and a potential flux through the TCA cycle.

[0077] Interestingly, while an increase in intracellular calcium was observed in the present studies, others have shown that HN suppresses the elevation of Ca²⁺ induced by Abeta (31-35) in a dose- and time-dependent manner. These effects observed here could be specific to mitochondria-rich islets. The ability of HNGF6A to increase intracellular calcium levels even in the presence of diazoxide is consistent with an effect on the augmentation phase and KATP channel-independent mechanism. In addition to ATP, effects of HN on the other metabolic couplers need to be explored to fully understand the mechanism through which HNGF6A increases insulin secretion.

[0078] An interesting observation is that the most insulin secreting effects of HN were seen in the presence of hyperglycemia. In the presence of no or basal glucose, the effects of HN on insulin secretion were modest (Fig 1A). This ability of HN to significantly increase insulin secretion only in the presence of high glucose has significant clinical implications. The risk of hypoglycemia is less and indeed that is what was observed when non-diabetic animals were treated with injections of HNGF6A (data not shown). GLP-1, similar to HN, has no effect on basal (3 mM glucose) insulin secretion) from islets isolated from Sprague Dawley rats. This is in contrast to the sulfonylurea glibenclamide, which induces insulin release at both 2.8 mM (~40 mg%, hypoglycemia) and 16.8 mM glucose thus increasing the risk of hypoglycemia.

[0079] HN increases insulin secretion in both wild type islets and db/db islets. 6 week treatment with HN has been shown earlier to decrease apoptosis, improve beta cell survival and glucose homeostasis in a rodent auto-immune model of diabetes. The effects demonstrated here are independent on the effects of HN on cell survival and apoptosis as evidenced by the short time course. Studies presented here demonstrate a novel role for HN on insulin secretion and link HN to mitochondrial metabolism.

[0080] Treatment with both HNGF6A and GLP-1 increases glucose-stimulated insulin secretion (GSIS) significantly, compared to either HNGF6A or GLP-1 alone. The increase in GSIS in response to combined treatment with HNGF6A and GLP-1 is associated with increases in ATP levels compared to either HNGF6A or GLP- 1 alone, linking mitochondrial metabolism to insulin secretion. REFERENCES

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Claims

What is claimed is:

1. A method of increasing insulin secretion in a subject comprising administering humanin or an analog of humanin to the subject in an amount effective to increase insulin secretion.

2. The method of claim 1, wherein insulin secretion is increased in the subject when blood glucose levels are elevated above normal.

3. The method of claim 1 or 2, wherein the subject has a reduced production of insulin prior to administration of the humanin or humanin analog.

4. The method of any of claims 1-3, wherein the subject has an age-associated defect in insulin secretion.

5. The method of any of claims 1-3, wherein the subject has hepatic steatosis.

6. The method of any of claims 1-3, wherein the subject has, or is at risk for, type-1 or type-2 diabetes mellitus or maturity onset diabetes of the young (MODY).

7. The method of any of claims 1-6, wherein the humanin analog comprises 17- 50 amino acids comprising the amino acid sequence of any of SEQ ID NO: l - SEQ ID NO:25.

8. The method of any of claims 1-6, wherein the humanin analog comprises 17- 26 amino acids comprising the amino acid sequence of any of SEQ ID NO: l - SEQ ID NO:25.

9. The method of any of claims 1-6, wherein the humanin analog comprises the amino acid sequence of any of SEQ ID NO: 1 - SEQ ID NO:25.

10. The method of any of claims 1-6, wherein the humanin analog consists of the amino acid sequence of any of SEQ ID NO:2 - SEQ ID NO:25.

1 1. The method of any of claims 1-6, wherein the humanin analog consists of the amino acid sequence of SEQ ID NO: 11.

12. The method of any of claims 1-1 1, wherein the humanin or humanin analog is administered by administering a vector encoding the humanin or humanin analog to the subject such that the humanin or humanin analog is expressed from the vector.

13. The method of any of claims 1-12, wherein glucagon-like peptide 1 (GLP-1) is administered to the subject in combination with humanin or an analog of humanin.

14. The method of any of claims 1-13, wherein the subject is a human.

15. A pharmaceutical composition comprising glucagon-like peptide 1 (GLP-1) and humanin or an analog of humanin in an amount effective to increase insulin secretion in a subject.

16. The pharmaceutical composition of claim 15, wherein the humanin analog comprises 17-50 amino acids comprising the amino acid sequence of any of SEQ ID NO: l - SEQ ID NO:25.

17. The pharmaceutical composition of claim 15, wherein the humanin analog comprises 17-26 amino acids comprising the amino acid sequence of any of SEQ ID NO: l - SEQ ID NO:25.

18. The pharmaceutical composition of claim 15, wherein the humanin analog comprises the amino acid sequence of any of SEQ ID NO: l - SEQ ID NO:25.

19. The pharmaceutical composition of claim 15, wherein the humanin analog consists of the amino acid sequence of any of SEQ ID NO:2 - SEQ ID NO:25.

20. The pharmaceutical composition of claim 15, wherein the humanin analog consists of the amino acid sequence of SEQ ID NO: 11.